IL-12-induced IFN-gamma is dependent on caspase-1 processing of the IL-18 precursor - PubMed (original) (raw)

IL-12-induced IFN-gamma is dependent on caspase-1 processing of the IL-18 precursor

G Fantuzzi et al. J Clin Invest. 1999 Sep.

Abstract

IL-12 and IL-18 are IFN-gamma-inducing cytokines. In the present study, the role of endogenous IL-18 in the induction of IFN-gamma by IL-12 was investigated in mice. In the presence of a specific inhibitor of caspase-1 (also known as IL-1beta-converting enzyme, or ICE) IL-12-induced IFN-gamma from splenocytes was reduced by 85%. Using splenocytes from ICE-deficient mice, IL-12-induced IFN-gamma was reduced by 80%. However, the role of ICE was not through processing and release of IL-1beta. Neutralizing anti-IL-18 IgG reduced IL-12-induced IFN-gamma in splenocytes by 85%. Splenocytes cultured in vitro spontaneously released IL-18 into the extracellular compartment over time. Extracellular levels of IL-18 significantly correlated with IL-12-induced IFN-gamma and were reduced in cells obtained from ICE-deficient mice. In vivo, IL-12 administration increased circulating levels of IL-18 in wild-type mice but not in ICE-deficient mice. Both neutralization of IL-18 and ICE deficiency significantly reduced induction of circulating IFN-gamma in mice receiving IL-12. The IL-18 precursor was constitutively expressed in the livers and spleens of untreated mice. Furthermore, administration of IL-12 significantly increased liver-associated IL-18 levels. These data demonstrate that endogenous, ICE-cleaved IL-18 significantly contributes to induction of IFN-gamma by IL-12.

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Figures

Figure 1

Figure 1

Effect of IL-18 neutralization on the induction of IFN-γ by IL-12 in vitro. Splenocytes were cultured for 48 hours with increasing concentrations of IL-12 (0.1–10 ng/mL) in the presence or absence of either neutralizing anti–IL-18 IgG or control IgG (50 μg/mL). IFN-γ levels were measured in the supernatants. Data are mean ± SEM of 3 mice per group and are representative of 1 experiment out of 3 performed. *P < 0.05, **P < 0.01 vs. RPMI or control IgG by repeated-measures ANOVA.

Figure 2

Figure 2

Effect of ICE inhibition on IFN-γ induced by IL-12 in vitro. Splenocytes were cultured for 48 hours with increasing concentrations of IL-12 (0.1–10 ng/mL) in the presence or absence of ICE inhibitor (20 μM). IFN-γ levels were measured in the supernatants. Data are mean ± SEM of 3 mice per group and are representative of 1 experiment out of 3 performed. *P < 0.05, **P < 0.01 by paired Student’s t test.

Figure 3

Figure 3

Production of IFN-γ in splenocytes from ICE KO mice. Splenocytes from WT or ICE KO mice were cultured for 48 hours with increasing concentrations of IL-12 (0.1–10 ng/mL). IFN-γ levels were measured in the supernatants. Data are mean ± SEM of 6 mice per group. **P < 0.01, ***P < 0.001 WT vs. ICE KO by unpaired Student’s t test.

Figure 4

Figure 4

IL-18 production in unstimulated and IL-12–stimulated splenocytes. Splenocytes were cultured in RPMI-FBS for increasing amounts of time without added exogenous stimulation, or with IL-12 at 10 ng/mL. Cell-associated and released IL-18 levels were measured. Data are mean ± SEM of 9 mice per group.

Figure 5

Figure 5

Release of IL-18 in splenocytes from ICE KO mice. Splenocytes from WT or ICE KO mice were cultured for 24 hours in the absence of any exogenous stimulation. IL-18 levels were measured in supernatants. Data are mean ± SEM of 8 mice per group. **P < 0.01 by factorial ANOVA.

Figure 6

Figure 6

Effect of in vivo administration of IL-12 on IFN-γ levels in ICE KO and WT mice. WT and ICE KO mice received 4 daily intraperitoneal injections of IL-12 (100 or 400 ng/mouse) or vehicle. Two hours after the fourth injection, blood was collected and serum was prepared for measurement of IFN-γ. Data are mean ± SEM of 10 mice per group. *P < 0.05 vs. WT mice by factorial ANOVA.

Figure 7

Figure 7

Increase in serum IL-18 levels after IL-12 administration. WT and ICE KO mice received 4 daily intraperitoneal injections of IL-12 (100 or 400 ng/mouse) or vehicle. Two hours after the fourth injection, blood was collected and serum was prepared. Data are mean ± SEM of 10 mice per group. *P < 0.05 vs. WT mice. ‡P < 0.01 vs. corresponding vehicle by factorial ANOVA.

Figure 8

Figure 8

Constitutive expression of pro–IL-18 in the spleens and livers on untreated mice. Spleen and liver homogenates from 3 untreated mice were subjected to Western blot analysis with a specific rabbit anti-murine IL-18 antiserum. Recombinant murine pro–IL-18 and mature IL-18 were used as standards.

Figure 9

Figure 9

Neutralization of IL-18 reduces IL-12–induced IFN-γ levels in vivo. Mice received 4 daily intraperitoneal injections of IL-12 (100 ng/mouse). One hour before the first and the third injection, the antibody-treated group received an intraperitoneal injection of 200 μL of rabbit anti–IL-18 antiserum, whereas the control group received the same amount of NRS. Two hours after the fourth injection, blood was collected and serum was prepared for measurement of IFN-γ. Data are mean ± SEM of 5 mice per group. **P < 0.01 vs. control by unpaired Student’s t test.

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